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Glycobiology Advance Access published online on October 24, 2008
Glycobiology, doi:10.1093/glycob/cwn116
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© The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

O-Fucosylation of Antibody Light Chain: Characterization of a Modification Occurring on an IgG1 Molecule

John F. Valliere-Douglass, Lowell J. Brady, Chris Farnsworth, Danielle Pace, Alain Balland, Alison Wallace, Wesley Wang, Michael J. Treuheit and Boxu Yan*

* Corresponding author: Email: valliere{at}amgen.com, meoweez{at}msn.com

Received on August 3, 2008; accepted on October 21, 2008

We describe the characterization of an O-fucosyl modification to a serine residue on the light chain of a recombinant, human IgG1 molecule expressed in Chinese hamster ovary (CHO) cells. Cation exchange (CEX) chromatography and hydrophobic interaction chromatography (HIC) were used to isolate a Fab population which was 146 Da heavier than the expected mass. Isolated Fab fragments were treated with reducing agent to facilitate mass spectrometric analysis of the reduced light chain (LC) and fragment difficult (Fd). Antibody light chain with a net addition of 146 Da was detected by mass spectrometric analysis of the modified Fab. A light chain tryptic peptide in complimentarity determining region-1 (CDR-1) was subsequently identified with a net addition of 146 Da by peptide map. Results from a nanospray infusion of the modified peptide into a linear ion trap mass spectrometer with electron transfer dissociation (ETD) functionality indicated that the modified residue was a serine at position 30 in the light chain. Acid hydrolysis of the modified tryptic peptide followed by fluorescent labeling with 2 aminoanthranilic acid (2AA) and HPLC comparison with monosaccharide standards confirmed the presence of fucose on the light chain peptide. The presence of O-fucose on an antibody has not been previously reported. Currently, O-fucose has been described as occurring on mammalian proteins with amino acid sequence motifs associated with epidermal growth factor (EGF) like repeats or thrombospondin type 1 repeats (TSR’s). The amino acid sequence around the modified Ser in the IgG1 molecule does not conform to any known O-fucosylation sequence motif and thus is the first description of this type of modification on a non-consensus sequence in a mammalian protein.

Key words: O-Fucosylation / Antibody / Light Chain / ETD Mass Spectrometry / Monosaccharide Analysis


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J. F. Valliere-Douglass, P. Kodama, M. Mujacic, L. J. Brady, W. Wang, A. Wallace, B. Yan, P. Reddy, M. J. Treuheit, and A. Balland
Asparagine-linked Oligosaccharides Present on a Non-consensus Amino Acid Sequence in the CH1 Domain of Human Antibodies
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